The present invention relates to novel crosslinked and substituted biocompatible polysaccharides, characterized by improved rheological properties and optionally exhibiting advantageous properties introduced by the substituents, such as, for example, a moisturizing or lipophilizing action, in comparison with a conventional crosslinked polysaccharide, and which can be used as biomaterials, in particular in the field of filler surgery, tissue repair or as articular material or fluids.
Numerous substituted and crosslinked polysaccharides are known from the prior art but none of the processes of the prior art has made it possible to obtain polysaccharides exhibiting the synergistically improved rheological properties according to the present invention.
EP 0 265 116 on behalf of FIDIA, describes crosslinked compounds substituted via ester functional groups on the —COOH functional groups of the polysaccharide. Specifically, the hyaluronic acid chains are crosslinked via ester bridges formed by intra- or intermolecular reaction between aliphatic polyalcohols and the carboxylic acid functional groups of the polysaccharide. Substitution also takes place via the ester functional groups of the polysaccharide by the grafting of small molecules carrying hydroxyl functional groups, such as ethanol or benzyl alcohol, for example.
In EP 0 341 745, also on behalf of FIDIA, the polymer is “self-crosslinked” in the sense that the ester functional groups are formed between the carboxyl functional groups of the polysaccharide and the hydroxyl functional groups of one and the same chain or of another chain, without crosslinking agent. The patent also discloses crosslinked and substituted polymers. The substituents are in this instance also alcohol chains, such as ethanol or benzyl alcohol, and are grafted to the polysaccharide via the carboxyl functional groups of the latter.
Patent application WO 99/43728, on behalf of FIDIA, reports hyaluronic acids sulfated in the N-position or O-position. These polysaccharides, which can, as in the abovementioned patent applications, be self-crosslinked or crosslinked via ester bridges (EP 0 256 116 and EP 0 341 745), are subsequently grafted with polyurethanes. A complex matrix of different cocrosslinked polymers is thus obtained. However, these products are also crosslinked via ester functional groups and furthermore require the use of at least three steps of synthesis in the case of a matrix of O- or N-sulfated polysaccharide and of polyurethane. Thus, they do not present a satisfactory solution.
Patent EP 0 749 982, on behalf of HERCULES, teaches the preparation of polymers substituted by hindered phenol compounds, with antioxidizing properties, which can optionally be crosslinked. The crosslinking is carried out by conventional means of the prior art using polyfunctional epoxides or corresponding halohydrins (U.S. Pat. No. 4,716,224, U.S. Pat. No. 4,863,907, EP 0 507 604 A2, U.S. Pat. No. 4,716,154, U.S. Pat. No. 4,772,419, U.S. Pat. No. 4,957,744), polyhydric alcohols (U.S. Pat. No. 4,582,865, U.S. Pat. No. 4,605,691), divinyl sulfone (U.S. Pat. No. 5,128,326, U.S. Pat. No. 4,582,865) and aldehydes (U.S. Pat. No. 4,713,448). According to the preferred embodiment, the polymer is crosslinked by reaction with carboxylic acids or polyacid anhydrides, in order to again result in the formation of esters. The compounds described in the prior art thus have a high density of ester functional groups in the majority of cases.
Cocrosslinked polymers are also known from the prior art, for example those described in patent application WO 2005/012364, on behalf of ANTEIS, which exhibit an improved persistence by the formation of a matrix by cocrosslinking of one or more polymers. These cocrosslinked polysaccharides can in addition be substituted by polymers having a low average molecular weight or small nonpolymeric molecules. These polymers are generally hyaluronic acid cocrosslinked with cellulose, to which compound will subsequently be grafted, via the crosslinking agent, with small polymers, such as heparin or hyaluronic acid, carrying benzyl esters (Mw<50 000 kDa). In some cases, antioxidants, such as vitamin C, are also grafted via the crosslinking agent.
U.S. Pat. No. 4,605,691, on behalf of Balazs (Biomatrix), also teaches a cocrosslinking process (a process of formation of cocrosslinked polymers). This time it concerns the cocrosslinking of hyaluronic acid with collagen, cellulose, heparin or carminic acid.
The common feature of the processes described in the prior art is that the substitutions or graftings which result in the functionalization take place via the crosslinking agent. In comparison with a crosslinking alone, this is reflected in a competition between reaction for crosslinking and functionalization via the crosslinking agent.
A person skilled in the art should thus always take care to properly adjust the amount of crosslinking agent introduced in order for the functionalization not to limit the crosslinking, which would result in a modification to the final properties of the gel according to the respective kinetics of the reactions.
Specifically, if the crosslinking takes place before the introduction of the functional agent, care will have to be taken, on the one hand, that the functional agent is introduced homogeneously into the crosslinked polymer network and, on the other hand, that there remains sufficient crosslinking agent to functionalize the polymer. If all is consumed, it is then necessary to add a further amount of crosslinking agent; the risk then arises of possible overcrosslinking. In some cases, a person skilled in the art will even have to substitute before crosslinking in order to limit the “competition” effects.
A person skilled in the art will thus still be confronted with a choice: to substitute before or after having crosslinked the polymer and to adjust the reaction conditions, taking into account the polymer/crosslinking agent and functionalization agent/crosslinking agent reaction rates. This choice thus becomes a crucial and problematic step of the process.
Water-soluble celluloses, such as sulfoalkylcelluloses substituted by sulfoalkyls and ethers, are also known. The desired aim is to obtain high functionalization in order to obtain water-soluble sulfoalkylcelluloses; see, for example, U.S. Pat. No. 4,990,609 on behalf of WOLFF WALSRODE. For this type of functionalization, sultones are often used for their high reactivity, which makes possible a high degree of functionalization and improves the solubility of the final product due to the strong presence of sulfonate functional groups. However, if it is desired to crosslink in a second step, the high degrees of substitution can interfere with the satisfactory crosslinking of the polymer. Furthermore, the functionalization conditions are often too drastic for the polysaccharide not to be damaged to the point of irredeemably diminishing its rheological properties; see, for example, the compounds described in U.S. Pat. No. 3,046,272.
This is because it is known that polysaccharides, for example hyaluronic acid, exhibit relatively poor resistance to alkaline conditions and it is known that, during the crosslinking or deacetylation of hyaluronic acid in sodium hydroxide solution, decomposition occurs (Simkovic et al., Carbohydrate Polymers, 41, 2000, 9-14); in point of fact, the reactions described in the prior art are often lengthy and/or under pH conditions which result in decomposition.
Under the highly concentrated alkaline conditions and at the high temperatures to which polysaccharides are subjected in processes, such as those described in U.S. Pat. No. 4,321,367 or in U.S. Pat. No. 4,175,183, it has been demonstrated (see the comparative examples below) that decomposition of the polysaccharide takes place.
Thus, in the processes described in the prior art, although relatively easy to carry out, the processes for producing a crosslinked and substituted polymer are very often lengthy and require several steps because of the successive addition of the various ingredients in order to avoid competition between the various polymers and grafts which will be attached to the polysaccharide via the crosslinking agent. Furthermore, due to the reaction times and the reaction conditions, the resulting polymer can have damaged rheological properties.
The present invention makes it possible to solve all of the disadvantages of the processes of the prior art and makes it possible in addition to obtain polysaccharides having rheological properties which are synergistically improved.
It relates to a process for the preparation of crosslinked and substituted polysaccharides. Principally, the crosslinking and substitution reactions in this process are carried out simultaneously, under the same experimental conditions and on the same reaction sites, the hydroxyl functional groups of the polysaccharide, this being the case without there being competition between the different entities involved, the substitutions not being carried out via the crosslinking agent. The degrees of substitution and crosslinking obtained are thus comparable to those obtained by reactions carried out sequentially and the rheological properties of the polysaccharides are improved.
The present invention also relates to a crosslinked and substituted polysaccharide obtained by the process according to the invention, the rheology of which, in particular the viscoelasticity of which, is increased in comparison not only with the simply substituted polysaccharide but also in comparison with the solely crosslinked polysaccharide and in comparison with the substituted and then crosslinked polysaccharide.
In addition, the substituents can introduce advantageous properties, for example biological properties, into the polysaccharide according to the invention, the rheological properties of which are improved. The synergistic effect is all the more surprising as it is retained during the sterilization of the substituted and crosslinked polysaccharide.
The present invention thus makes it possible to combine the advantages relating to the substitution and those relating to the crosslinking without modifying the individual characteristics of each of these modifications taken separately and in particular without damaging the rheological properties since they are synergistically improved.
The invention relates to a process for the simultaneous substitution and crosslinking of a polysaccharide via its hydroxyl functional groups, in an aqueous phase, comprising the following steps:                a polysaccharide is placed in an aqueous medium,        it is brought into the presence of at least one precursor of a substituent,        it is brought into the presence of a crosslinking agent,        the substituted and crosslinked polysaccharide is obtained and isolated, wherein, said process is carried out in the presence of a basic or acidic catalyst, the concentration of which is between 3.16×10−7 and 0.32 mol/L, and at a temperature of less than 60° C.        
The term “via its hydroxyl functional groups” is understood to mean the fact that the substitutions and the crosslinkings are carried out on the —OH groups carried by the polysaccharides.
The process can also be characterized by a reactive catalyst ratio or RCR.
This reactive catalyst ratio (RCR) is defined as being:
      R    ⁢                  ⁢    C    ⁢                  ⁢    R    =                                          (                          Number              ⁢                                                          ⁢              of              ⁢                                                          ⁢              moles              ⁢                                                          ⁢              reactive              ⁢                                                          ⁢              functional              ⁢                                                          ⁢              groups              ⁢                                                          ⁢              of                                                                                                              ⁢                          the              ⁢                                                          ⁢              catalyst              ⁢                                                          ⁢              introduced              ⁢                                                          ⁢              into              ⁢                                                          ⁢              the              ⁢                                                          ⁢              reaction              ⁢                                                          ⁢              medium                        )                                                                    (                          Number              ⁢                                                          ⁢              of              ⁢                                                          ⁢              moles              ⁢                                                          ⁢              disacharide              ⁢                                                          ⁢              unit                                                                                      introduced              ⁢                                                          ⁢              into              ⁢                                                          ⁢              the              ⁢                                                          ⁢              reaction              ⁢                                                          ⁢              medium                        )                              
In one embodiment, the reactive catalyst ratio in the process according to the invention is between 0.02:1 and 3:1.
In one embodiment, the reactive catalyst ratio in the process according to the invention is between 0.2:1 and 3:1.
In one embodiment, the reactive catalyst ratio in the process according to the invention is between 0.3:1 and 3:1.
In one embodiment, the reactive catalyst ratio in the process according to the invention is between 0.5:1 and 2:1.
In one embodiment, the reactive catalyst ratio in the process according to the invention is between 0.7:1 and 1.5:1.
In one embodiment, the reactive catalyst ratio in the process according to the invention is 1.75:1.
In one embodiment, the reactive catalyst ratio in the process according to the invention is 1:1.
In one embodiment, the reactive catalyst ratio in the process according to the invention is 0.8:1.
In one embodiment, the reactive catalyst ratio in the process according to the invention is 0.06:1.
In one embodiment, the catalyst in the process according to the invention is a base.
In this embodiment, the reactive functional group of the catalyst is the HO− ion.
In the aqueous phase, the situation is that pH=14+log([HO−]) and [HO−]=10−(14-pH).
In one embodiment, the concentration of catalyst HO− in the process according to the invention is between 10−6 mol/L and 0.32 mol/L, such that 10−6 mol/L≦[HO−]≦0.32 mol/L.
In one embodiment, the concentration of catalyst HO− in the process according to the invention is between 3.16×10−4 mol/L and 3.16×10−2 mol/L, such that 3.16×10−4 mol/L≦[HO−]≦3.16×10−2 mol/L.
In one embodiment, the catalyst in the process according to the invention is an inorganic base.
In one embodiment, the inorganic base in the process according to the invention is chosen from the group consisting of soda (sodium hydroxide) or potash (potassium hydroxide).
In one embodiment, the concentration by weight of the inorganic base in the process according to the invention is between 1.2×10−5% and 1.3%.
In one embodiment, the concentration by weight of the inorganic base in the process according to the invention is between 0.25% and 1.1%.
In one embodiment, the concentration by weight of the inorganic base in the process according to the invention is 1%.
In one embodiment, the concentration by weight of the inorganic base in the process according to the invention is 0.5%.
In one embodiment, the catalyst in the process according to the invention is an organic base.
In one embodiment, the organic base in the process according to the invention is pyridine.
In one embodiment, the pH of the aqueous reaction medium in the process according to the invention is basic.
In one embodiment, the pH of the aqueous reaction medium in the process according to the invention is within a range from 8 to 13.5.
In one embodiment, the pH of the aqueous reaction medium in the process according to the invention is within a range from 10.5 to 12.5.
In one embodiment, the catalyst in the process according to the invention is an acid.
In this embodiment, the reactive functional group of the catalyst is the H3O+ ion.
In one embodiment, the concentration of catalyst H3O+ in the process according to the invention is between 3.16×10−7 mol/L and 0.01 mol/L, such that 3.16×10−7 mol/L≦[H3O−]≦0.01 mol/L.
In one embodiment, the concentration of catalyst H3O+ in the process according to the invention is between 10−6 mol/L and 3.16×10−5 mol/L, such that 10−6 mol/L≦[H3O+]≦3.16×10−5 mol/L.
In the aqueous phase, the situation is that pH=−log([H3O+]) and [H3O+]=10−pH.
In one embodiment, the acid in the process according to the invention is an inorganic acid.
In one embodiment, the inorganic acid in the process according to the invention is hydrochloric acid.
In one embodiment, the concentration by weight of the inorganic acid in the process according to the invention is between 1.14×10−5% and 1.15%.
In one embodiment, the concentration by weight of the inorganic acid in the process according to the invention is between 0.05% and 1%.
In one embodiment, the concentration by weight of the inorganic acid in the process according to the invention is between 0.05% and 0.36%.
In one embodiment, the catalyst in the process according to the invention is an organic acid.
In one embodiment, the organic acid is chosen from the group consisting of glutamic acid and acetic acid.
In one embodiment, the concentration by weight of the organic acid in the process according to the invention is between 0.25% and 2%.
In one embodiment, the concentration by weight of the organic acid in the process according to the invention is between 0.25% and 1.1%.
In one embodiment, the concentration by weight of the organic acid in the process according to the invention is 1%.
In one embodiment, the pH of the aqueous reaction medium in the process according to the invention is acidic.
In one embodiment, the pH of the aqueous reaction medium in the process according to the invention is within a range from 2 to 6.5.
In one embodiment, the pH of the aqueous reaction medium in the process according to the invention is within a range from 4.5 to 6.
The basic or acidic catalyst is soluble in an aqueous medium.
In one embodiment, the polysaccharide in the process according to the invention is chosen from the group consisting of hyaluronic acid or one of its salts, chitosan, cellulose and their derivatives.
In one embodiment, the polysaccharide is hyaluronic acid.
In one embodiment, the polysaccharide is sodium hyaluronate.
In one embodiment, the polysaccharide is chitosan.
In one embodiment, the chitosan is partially deacetylated.
In one embodiment, a chitosan with a degree of deacetylation of approximately 80% is used.
In one embodiment, the polysaccharide is cellulose or one of its derivatives.
In one embodiment, the polysaccharide is carboxymethylcellulose.
The term Mw or “molecular weight” is used to describe the weight-average molecular weight of the polysaccharide, measured in daltons.
In one embodiment, the molecular weight of the polysaccharide is within a range from 0.01 MDa to 4.0 MDa.
In one embodiment, the molecular weight of the polysaccharide is within a range from 0.1 MDa to 3.6 MDa.
In one embodiment, the molecular weight of the polysaccharide is within a range from 0.10 MDa to 0.15 MDa.
In one embodiment, the molecular weight of the polysaccharide is within a range from 0.9 MDa to 2 MDa.
In one embodiment, the molecular weight of the polysaccharide is within a range from 2.5 to 3.6 MDa.
In one embodiment, the molecular weight Mw of the polysaccharide is 2.7 MDa.
In one embodiment, the molecular weight Mw of the polysaccharide is 1.5 MDa.
In one embodiment, the molecular weight Mw of the polysaccharide is 1.0 MDa.
In one embodiment, the molecular weight Mw of the polysaccharide is 120 000 Da.
In one embodiment, the crosslinking agent in the process according to the invention is bi- or polyfunctional.
In one embodiment, the bi- or polyfunctional crosslinking agent in the process according to the invention has at least one epoxide functional group.
In one embodiment, the bi- or polyfunctional crosslinking agent in the process according to the invention is chosen from the group consisting of ethylene glycol diglycidyl ether, butanediol diglycidyl ether, polyglycerol polyglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, a bisepoxy or a polyepoxy, such as 1,2,3,4-diepoxybutane or 1,2,7,8-diepoxyoctane.
In one embodiment, the bi- or polyfunctional crosslinking agent in the process according to the invention is epichlorohydrin.
In one embodiment, the bi- or polyfunctional crosslinking agent in the process according to the invention has at least one vinyl functional group.
In one embodiment, the bi- or polyfunctional crosslinking agent in the process according to the invention is a dialkyl sulfone wherein the identical or different and linear or branched alkyl groups are chains having from 1 to 4 carbon atoms.
In one embodiment, the bi- or polyfunctional crosslinking agent in the process according to the invention is divinyl sulfone.
In one embodiment, the crosslinking agent in the process according to the invention is a mono-, bi- or polyaldehyde.
In one embodiment, the crosslinking agent in the process according to the invention is formaldehyde.
In one embodiment, the crosslinking agent in the process according to the invention is glutaraldehyde.
In one embodiment, the molar ratio of the crosslinking agent to the polysaccharide employed in the process according to the invention is within a range from 0.001 to 0.5.
In one embodiment, the molar ratio of the crosslinking agent to the polysaccharide employed in the process according to the invention is within a range from 0.01 to 0.3.
In one embodiment, the molar ratio of the crosslinking agent to the polysaccharide employed in the process according to the invention is within a range from 0.05 to 0.2.
In one embodiment, the molar ratio of the crosslinking agent to the polysaccharide employed in the process according to the invention is equal to 0.07.
In one embodiment, the molar ratio of the crosslinking agent to the polysaccharide employed in the process according to the invention is equal to 0.08.
In one embodiment, the molar ratio of the crosslinking agent to the polysaccharide employed in the process according to the invention is equal to 0.10.
In one embodiment, the molar ratio of the crosslinking agent to the polysaccharide employed in the process according to the invention is equal to 0.14.
In one embodiment, the molar ratio of the crosslinking agent to the polysaccharide employed in the process according to the invention is equal to 0.21.
In one embodiment, the precursor of the substituent in the process according to the invention is chosen from the group of the molecules comprising just one reactive functional group chosen from the group consisting of vinyl, epoxide, allyl, ketone, aldehyde, thiocyanate, halide, isocyanate, halosilicon, nitrile and sultone functional groups.
The term “reactive functional group” is understood to mean a functional group capable of forming a bond with a hydroxyl functional group of the polysaccharide.
In one embodiment, the bond is formed by creation of an ether bond.
In one embodiment, the bond is formed by creation of a hemiacetal bond.
In one embodiment, the bond is formed by the creation of a urethane bond.
Under the conditions of the process according to the invention, the formation of an ester functional group is thus excluded.
In one embodiment, the precursor of the substituent in the process according to the invention is chosen from the group consisting of molecules additionally comprising at least one advantageous functional group or group, inert with regard to the substitution and crosslinking reactions, chosen from the group consisting of sulfonate, linear or branched alkyl, substituted or unsubstituted aromatic, sulfate, thiol, monosaccharide, phosphate, phosphonate, carbonate and ester groups or functional groups.
The term “inert” is understood to mean a functional group which does not react under the conditions of implementation of the process and which is stable under the conditions of storage of the product obtained according to the process of the invention. A functional group which, under the conditions of implementation of the process, would be capable of not reacting with any of the functional groups of the polysaccharide or with any of the functional groups of the crosslinking agent or with any of the reactive functional groups of the precursor of the substituent is thus inert under the conditions of implementation of the process.
In one embodiment, the precursor of the substituent in the process according to the invention is chosen from the group consisting of the molecules of general formula F—R-(G)x, wherein:                F is a reactive functional group chosen from the group consisting of substituted or unsubstituted vinyl, substituted or unsubstituted epoxide, substituted or unsubstituted allyl, ketone, aldehyde, thiocyanate, halide, isocyanate, halosilicon, nitrile and sultone functional groups; R is a bond or an alkyl chain having from 1 to 12 carbon atoms, linear or branched, substituted or unsubstituted aromatic, saturated or unsaturated, optionally comprising one or more heteroatoms;        G is either a hydrogen or an advantageous functional group or group, which are inert, chosen from the group consisting of the sulfonate, linear or branched alkyl, substituted or unsubstituted aromatic, sulfate, thiol, monosaccharide, phosphate, phosphonate, carbonate and ester groups or functional groups;        x is a natural integer such that 1≦x≦3.        
In one embodiment, F in the process according to the invention is a vinyl functional group and the precursor of the substituent is chosen from the group consisting of compounds of formula:
                R and G being as defined above,        R1, R2 and R3, which are identical or different, being either a hydrogen atom or an alkyl chain having from 1 to 3 carbon atoms.        
In one embodiment, F in the process according to the invention is an epoxide functional group and the precursor of the substituent is chosen from the group consisting of compounds of formula:
                R and G being as defined above,        R1, R2 and R3 being as defined above.        
In one embodiment, F in the process according to the invention is an allyl functional group and the precursor of the substituent is chosen from the group consisting of compounds of formula:
                R and G being as defined above,        R1, R2 and R3 being as defined above,        R4 and R5, which are identical or different, being either a hydrogen atom or an alkyl chain having from 1 to 3 carbon atoms.        
In one embodiment, G in the process according to the invention is a sulfate functional group.
In one embodiment, G in the process according to the invention is a hydrogen atom.
In one embodiment, G in the process according to the invention is a sulfonate functional group.
In one embodiment, the precursor of the substituent in the process according to the invention is chosen from the group consisting of allyl-sulfates, epoxy-sulfates, vinyl-sulfonates and epoxy-alkanes.
In one embodiment, the substituent in the process according to the invention is chosen from the group consisting of vinylsulfonic acid and its salts, epoxybutane and sodium allyl sulfate.
In one embodiment, the polyfunctional crosslinking agent in the process according to the invention is 1,4-butanediol diglycidyl ether (BDDE) and the precursor of the substituent is sodium vinylsulfonate.
In one embodiment, the polyfunctional crosslinking agent in the process according to the invention is 1,4-butanediol diglycidyl ether (BDDE) and the precursor of the substituent is sodium allyl sulfate.
In one embodiment, the polyfunctional crosslinking agent in the process according to the invention is 1,4-butanediol diglycidyl ether (BDDE) and the precursor of the substituent is epoxybutane.
In one embodiment, the polyfunctional crosslinking agent in the process according to the invention is divinyl sulfone and the precursor of the substituent is sodium vinylsulfonate.
In one embodiment, the molar ratio of the precursor of the substituent to the polysaccharide employed in the process according to the invention is within a range from 0.001 to 4.00.
In one embodiment, the molar ratio of the precursor of the substituent to the polysaccharide employed in the process according to the invention is within a range from 0.20 to 2.20.
In one embodiment, the molar ratio of the precursor of the substituent to the polysaccharide employed in the process according to the invention is equal to 0.24.
In one embodiment, the molar ratio of the precursor of the substituent to the polysaccharide employed in the process according to the invention is equal to 0.30.
In one embodiment, the molar ratio of the precursor of the substituent to the polysaccharide employed in the process according to the invention is equal to 0.35.
In one embodiment, the molar ratio of the precursor of the substituent to the polysaccharide employed in the process according to the invention is equal to 0.90.
In one embodiment, the molar ratio of the precursor of the substituent to the polysaccharide employed in the process according to the invention is equal to 1.00.
In one embodiment, the molar ratio of the precursor of the substituent to the polysaccharide employed in the process according to the invention is equal to 1.60.
In one embodiment, the molar ratio of the precursor of the substituent to the polysaccharide employed in the process according to the invention is equal to 2.00.
Principally, the crosslinking and substitution reactions in the process according to the invention are carried out simultaneously, under the same experimental conditions and on the same reaction sites of the polysaccharide, this being the case without there being competition between the various entities involved. The degrees of substitution and crosslinking are the same as those of the reactions carried out in isolation.
The process thus makes it possible to be able to control the crosslinking independently of the substitution in order to facilitate the preparation of the gels and to be able to easily adapt the product according to the use thereof.
The present invention, because the substitution and the crosslinking are simultaneous, makes it possible to limit the time during which the polysaccharide is present in an alkaline medium which decomposes it if residence is prolonged. Specifically, it is well known that, in the case of a simple substitution in an alkaline medium, for example, hyaluronic acid is rapidly decomposed and loses all its gelling and viscoelastic properties. A surprising effect of the invention is that the fact that the crosslinking and substitution reactions are simultaneous protects the polysaccharide during the reaction and makes it possible to obtain a synergistic effect with regard to the rheological properties, in particular the elasticity of the polysaccharide, which is greatly increased.
The advantages related to the fact that the crosslinking/substitution reactions are simultaneous are not limited to advantages visible on the final product, such as a better elasticity, a good homogeneity of the gel, homogeneous distribution of the substituents or the limitation of the decomposition of the polysaccharide during the crosslinking/substitution, but also comprise the reaction time and in particular the number of reaction steps. The process of the invention makes possible simultaneous introduction of all the reactants. Just one single reaction step offers not only a considerable saving in time but also limits losses of time and of solvents. As all the reactions involved, namely crosslinking and substitution reactions, take place under the same conditions, the catalyst introduced will be active for both reactions without it being necessary to increase its amount in comparison with a simple crosslinking. The absence of competition between crosslinking and substitution prevents it from being necessary to add an excess of reactant in order to compensate for the decomposition thereof or the excessively rapid consumption thereof.
The process of the invention does not use catalysts other than simple acids or bases, does not use organic solvents and does not use activating agents, and the atomic balance of the reaction is good, given the absence of formation of byproducts.
The latter point is the other advantage of the invention: the process of the invention does not produce byproducts which have to be removed during the purification. It is simply a matter of rinsing the product in order to remove the excess crosslinking agent and catalyst. In view of the applications of the polysaccharides obtained, in particular as biomaterials, this absence of byproducts is a real competitive advantage.
The process which makes it possible to obtain the compounds of the present invention differs from the prior art in that the simple implementation thereof makes it possible, surprisingly, to substitute and crosslink a polysaccharide simultaneously without the substitution being in competition with the crosslinking. Better control is thus exercised over the degree of crosslinking or the degree of substitution.
The process which makes it possible to obtain the products of the present invention offers complete freedom with regard to the parametering of each of the reactions occurring simultaneously and independently in the reaction medium. It is thus possible to modify the degree of crosslinking without influencing the substitution and, conversely, it is possible to modify the degree of substitution without influencing the crosslinking.
The implementation of the crosslinking process of the invention makes it possible to obtain a product of high homogeneity which can be easily injected. Surprisingly, the process according to the invention makes it possible to enhance the rheological properties of the crosslinked polysaccharides without having to employ more crosslinking agent. In addition, it makes it possible to introduce other properties, such as hydration or lipophilicity.
The process of the invention is such that it is possible to envisage carrying out up to three reactions simultaneously. The term “three simultaneous reactions” is understood to mean a crosslinking simultaneously with a double substitution.
In one embodiment, the polysaccharide obtained by the process according to the invention is substituted on its hydroxyl functional groups.
The degree of substituent introduced (DSI) is defined as being:
      D    ⁢                  ⁢    S    ⁢                  ⁢    I    =                                          (                          Number              ⁢                                                          ⁢              of              ⁢                                                          ⁢              moles              ⁢                                                          ⁢              of              ⁢                                                          ⁢              reactive              ⁢                                                          ⁢              fonctional              ⁢                                                          ⁢              groups              ⁢                                                          ⁢              of                                                                                      the              ⁢                                                          ⁢              substituent              ⁢                                                          ⁢              introduced              ⁢                                                          ⁢              into              ⁢                                                          ⁢              the              ⁢                                                          ⁢              reaction              ⁢                                                          ⁢              medium                        )                                                                    (                          Number              ⁢                                                          ⁢              of              ⁢                                                          ⁢              moles              ⁢                                                          ⁢              of              ⁢                                                          ⁢              disaccharide              ⁢                                                          ⁢              unit                                                                                      introduced              ⁢                                                          ⁢              into              ⁢                                                          ⁢              the              ⁢                                                          ⁢              reaction              ⁢                                                          ⁢              medium                        )                              
In one embodiment, the degree of substituent introduced in the process according to the invention is within a range from 0.001 to 4.00 (0.001≦DSI≦4.00).
In one embodiment, the degree of substituent introduced in the process according to the invention is within a range from 0.20 to 2.20 (0.20≦DSI≦2.20).
In one embodiment, the degree of substituent introduced in the process according to the invention is equal to 0.24.
In one embodiment, the degree of substituent introduced in the process according to the invention is equal to 0.30.
In one embodiment, the degree of substituent introduced in the process according to the invention is equal to 0.35.
In one embodiment, the degree of substituent introduced in the process according to the invention is equal to 0.90.
In one embodiment, the degree of substituent introduced in the process according to the invention is equal to 1.00.
In one embodiment, the degree of substituent introduced in the process according to the invention is equal to 1.60.
In one embodiment, the degree of substituent introduced in the process according to the invention is equal to 2.00.
In one embodiment, the polysaccharide obtained in the process according to the invention is crosslinked by the reaction of the crosslinking agent with its hydroxyl functional groups.
The degree of crosslinking agent introduced (DCI) is defined as being:
      D    ⁢                  ⁢    C    ⁢                  ⁢    I    =                                          (                          Number              ⁢                                                          ⁢              of              ⁢                                                          ⁢              moles              ⁢                                                          ⁢              crosslinking              ⁢                                                          ⁢              agent                                                                                      introduced              ⁢                                                          ⁢              into              ⁢                                                          ⁢                              t                ⁢                he                            ⁢                                                          ⁢              reaction              ⁢                                                          ⁢              medium                        )                                                                    (                          Number              ⁢                                                          ⁢              of              ⁢                                                          ⁢              moles              ⁢                                                          ⁢              disacharide              ⁢                                                          ⁢              unit                                                                                      introduced              ⁢                                                          ⁢              into              ⁢                                                          ⁢              the              ⁢                                                          ⁢              reaction              ⁢                                                          ⁢              medium                        )                              
In one embodiment, the degree of crosslinking agent introduced in the process according to the invention is within a range from 0.001 to 0.5.
In one embodiment, the degree of crosslinking agent introduced in the process according to the invention is within a range from 0.01 to 0.3.
In one embodiment, the degree of crosslinking agent introduced in the process according to the invention is 0.07.
In one embodiment, the degree of crosslinking agent introduced in the process according to the invention is 0.08.
In one embodiment, the degree of crosslinking agent introduced in the process according to the invention is 0.10.
In one embodiment, the degree of crosslinking agent introduced in the process according to the invention is 0.14.
In one embodiment, the degree of crosslinking agent introduced in the process according to the invention is 0.21.
In one embodiment, the ratio of the weight of polysaccharide employed to the weight of water employed in the process according to the invention is within a range from 4% to 20%, as percentage by weight.
In one embodiment, the ratio of the weight of polysaccharide employed to the weight of water employed in the process according to the invention is within a range from 6% to 16%, as percentage by weight.
In one embodiment, the ratio of the weight of polysaccharide employed to the weight of water employed in the process according to the invention is within a range from 8% to 14%, as percentage by weight.
In one embodiment, the ratio of the weight of polysaccharide employed to the weight of water employed in the process according to the invention is 5.7%, as percentage by weight.
In one embodiment, the ratio of the weight of polysaccharide employed to the weight of water employed in the process according to the invention is 6.3%, as percentage by weight.
In one embodiment, the ratio of the weight of polysaccharide employed to the weight of water employed in the process according to the invention is 10.3%, as percentage by weight.
In one embodiment, the ratio of the weight of polysaccharide employed to the weight of water employed in the process according to the invention is 11.1%, as percentage by weight.
In one embodiment, the ratio of the weight of polysaccharide employed to the weight of water employed in the process according to the invention is 12.2%, as percentage by weight.
In one embodiment, the ratio of the weight of polysaccharide employed to the weight of water employed in the process according to the invention is 13.5%, as percentage by weight.
In one embodiment, the ratio of the weight of polysaccharide employed to the weight of water employed in the process according to the invention is 14.3%, as percentage by weight.
In one embodiment, the ratio of the weight of polysaccharide employed to the weight of water employed in the process according to the invention is 15.8%, as percentage by weight.
In one embodiment, the process according to the invention is carried out at room temperature.
The term “room temperature” is understood to mean a temperature between 18° C. and 25° C.
In one embodiment, the process according to the invention is carried out at a temperature of greater than 25° C.
In one embodiment, the process according to the invention is carried out at a temperature of less than 60° C.
In one embodiment, the process according to the invention is carried out at a temperature of between 39° C. and 60° C.
In one embodiment, the process according to the invention is carried out at a temperature of 40° C.
In one embodiment, the process according to the invention is carried out at a temperature of 50° C.
In one embodiment, the process according to the invention is carried out for a period of time within a range from 15 minutes to 48 hours.
In one embodiment, the process according to the invention is carried out for a period of time of 1 to 2 hours.
In one embodiment, the process according to the invention is carried out for a period of time of 2 to 3 hours.
In one embodiment, the process according to the invention is carried out for a period of time of 3 to 4 hours.
In one embodiment, the process according to the invention is carried out for a period of time of 4 to 5 hours.
In one embodiment, the process according to the invention additionally comprises a step of washing the polysaccharide obtained.
In one embodiment, the process according to the invention additionally comprises a step of washing the polysaccharide obtained with a buffer solution having a pH of approximately 7.
In one embodiment, the process according to the invention additionally comprises a step of washing the polysaccharide obtained with purified water.
In one embodiment, the crosslinking of the polysaccharide in the product obtained according to the process of the invention is carried out by dialiyldialkyl sulfone bridges of formula PS—O—(CH2)n-S(O2)-(CH2)n-O—PS, where “PS” represents the polysaccharide residue and n represents an integer such that 1≦n≦4.
In one embodiment, the crosslinking of the polysaccharide in the product obtained according to the process of the invention is carried out by diethyl sulfone bridges of formula PS—O—CH2—CH2—S(O2)—CH2—CH2—O—PS.
In one embodiment, the crosslinking of the polysaccharide in the product obtained according to the process of the invention is carried out by bridges of formula PS—O—CH2—CH(OH)—CH2—X—CH2—CH(OH)—CH2—O—PS, the X group being either an alkyl chain having from 2 to 6 carbon atoms or a polyether chain.
In one embodiment, the crosslinking of the polysaccharide in the product obtained according to the process of the invention is carried out by ether bridges of formula PS—O—CH2—O—PS.
In one embodiment, the crosslinking of the polysaccharide in the product obtained according to the process of the invention is carried out by hemiacetal bridges of formula PS—O—CH(OH)—(CH2)m—CH(OH)—O—PS, where m is an integer such that 0≦m≦4.
In one embodiment, the polysaccharide in the product obtained according to the process of the invention carries, on at least one of its hydroxyl functional groups, at least one substituent resulting from vinylsulfonic acid, 2-ethoxyethylsulfonic acid.
In one embodiment, the polysaccharide in the product obtained according to the process of the invention carries, on at least one of its hydroxyl functional groups, at least one substituent resulting from epoxybutane, 1-ethoxybutan-2-ol.
In one embodiment, the polysaccharide in the product obtained according to the process of the invention carries, on at least one of its hydroxyl functional groups, at least one substituent resulting from sodium allyl sulfate, sodium 3-propoxy sulfate.
The invention relates to the use of a hydrogel obtained according to the process of the invention in the formulation of a viscosupplementation composition.
The process according to the invention also relates to the compositions comprising a polysaccharide obtained by the process according to the invention.
In one embodiment, the polysaccharide obtained by the process according to the invention is in the gel or hydrogel form.
On conclusion of the crosslinking and substitution, it may be advantageous to neutralize the gel obtained according to standard processes known in the field, for example by addition of acid, when the process is carried out in a basic medium, and by addition of a base, when the process is carried out in an acidic medium.
The mixture obtained on conclusion of the process of the invention can optionally be subjected to an additional hydration step, in order to obtain a gel in the form of an injectable hydrogel suitable for the applications envisaged.
This hydration is generally carried out, in an aqueous medium, by simple mixing of the crosslinked and substituted gel with an aqueous solution, advantageously a buffered physiological aqueous solution, so as to obtain a final concentration which can vary within very wide proportions, according to the nature of the polysaccharides used, according to their respective degrees of crosslinking and also according to the use envisaged. The buffered solution which can be used can, for example, be an iso-osmolar physiological solution exhibiting a pH of between approximately 6.8 and approximately 7.5.
This final concentration of total polysaccharides is generally between approximately 5 and approximately 100 mg/g, preferably between approximately 5 and approximately 50 mg/g, for example approximately 20 mg/g, of hydrogel.
The invention relates to the use of a polysaccharide obtained according to the process of the invention in the formulation of a viscosupplementation composition.
The invention relates to the use of a polysaccharide obtained according to the process of the invention in the formulation of a composition for filling in wrinkles.
The applications targeted are more particularly the applications commonly observed in the context of injectable polysaccharide viscoelastic products used or which can potentially be used in the following pathologies or treatments:                cosmetic injections: for filling in wrinkles, skin defects or defects Of volume (cheekbones, chins, lips);        treatment of osteoarthritis, injection into the joint to replace or supplement deficient synovial fluid;        periurethral injection in the treatment of urinary incontinence by sphincter insufficiency;        postsurgical injection for preventing peritoneal adhesions in particular;        injection subsequent to surgery for far-sightedness by scleral incisions using a laser;        injection into the vitreous cavity.        
More particularly, in cosmetic surgery, according to its viscoelastic properties and properties of persistence, the hydrogel obtained according to the process of the invention can be used:                for filling in fine, moderate or deep wrinkles and can be injected with thin needles (27-gauge, for example);        as volumizing product with injection via needles with a larger diameter, for example from 22- to 26-gauge, and with a greater length (30 to 40 mm, for example); in this case, its cohesive nature will make it possible to guarantee that it is maintained at the site of the injection.        
The polysaccharide obtained according to the process of the invention also has an important application in joint surgery and in dental surgery for filling in periodontal pockets, for example.
These implementational examples are in no way limiting, the polysaccharide obtained according to the process of the present invention being more widely provided for:                filling in volumes;        generating spaces within certain tissues, thus promoting their optimum functioning;        replacing deficient physiological fluids.        
The polysaccharide obtained according to the process of the invention also has an application in the preparation of bone substitutes.
The polysaccharide obtained according to the process of the invention can also have an entirely advantageous application as matrix for releasing one (or more) active principle(s) dispersed beforehand within it. The term “active principle” is understood to mean any product which is active pharmacologically: medicinal active principle, antioxidant active principle (sorbitol, mannitol, and the like), antiseptic active principle, anti-inflammatory active principle, local anesthetic active principle (lidocaine, and the like), and the like.
In practice, the polysaccharide obtained according to the process of the invention, preferably after purification and hydration to give the hydrogel, can be packaged, for example in syringes, and sterilized according to any means known per se (for example by autoclaving) in order to be sold and/or used directly.
According to another aspect, the present invention relates to a kit comprising a polysaccharide obtained according to the process of the invention packaged in a sterile syringe.
The characteristics of the polysaccharides obtained according to the process of the invention are demonstrated in the examples below.